Establishment of embryonic stem cells secreting human factor VIII for cell-based treatment of hemophilia A

Summary.  Background:  Hemophilia A is an X-chromosome-linked recessive bleeding disorder resulting from an F8 gene abnormality. Although various gene therapies have been attempted with the aim of eliminating the need for factor VIII replacement therapy, obstacles to their clinical application remain. Objectives:  We evaluated whether embryonic stem (ES) cells with a tetracycline-inducible system could secrete human FVIII. Methods and results:  We found that embryoid bodies (EBs) developed under conditions promoting liver differentiation efficiently secreted human FVIII after doxycycline induction. Moreover, use of a B-domain variant F8 cDNA (226aa/N6) dramatically enhanced FVIII secretion. Sorting based on green fluorescent protein (GFP)–brachyury (Bry) and c-kit revealed that GFP–Bry⁺/c-kit⁺ cells during EB differentiation with serum contain an endoderm progenitor population. When GFP–Bry⁺/c-kit⁺ cells were cultured under the liver cell-promoting conditions, these cells secreted FVIII more efficiently than other populations tested. Conclusion:  Our findings suggest the potential for future development of an effective ES cell-based approach to treating hemophilia A.